2.4. Metabolic Stability Assay

Microsomal stability. The in vitro microsomal stability assay was conducted in triplicate in mouse and human liver microsomal systems, which were supplemented with nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor. Briefly, a compound (1 µM final concentration) was spiked into the reaction mixture containing liver microsomal protein (0.5 mg/mL final concentration) and MgCl₂ (1 mM final concentration) in 0.1 M potassium phosphate buffer (pH 7.4). The reaction was initiated by addition of 1 mM NADPH, followed by incubation at 37 °C. A negative control was performed in parallel without NADPH to reveal any chemical instability or non-NADPH dependent enzymatic degradation for each compound. At various time points (0, 5, 15, 30 and 60 min), 1 volume of reaction aliquot was taken and quenched with 3 volumes of acetonitrile containing 0.1% formic acid. The samples were then vortexed and centrifuged at 15,000 rpm for 5 min at 4 °C. The supernatants were collected and analyzed by LC/MS/MS to determine the remaining percentage and in vitro metabolic half-life (t_1/2).